Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 μM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.     

MATE2 mediates vacuolar sequestration of flavonoid glycosides and glycoside malonates in Medicago truncatula

The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3'-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage.     

褐色种皮大豆与其黄色种皮衍生亲本的表型及基因型比较

大豆种皮色在从野生大豆到栽培大豆的选择过程中逐渐由黑色变成黄色,是重要的形态标记,因此,大豆种皮色相关基因的研究无论是对进化理论研究还是育种实践都具有非常重要的意义。利用褐色种皮J1265-2大豆及其衍生亲本黄色种皮大豆J1265-1为材料,通过SSR引物扩增片段,检验遗传背景的异同,同时对控制种皮的候选基因GmF3’H进行扩增和测序分析。结果表明,褐色种皮和黄色种皮材料不仅用161对SSR分子标记检测没有发现差异,其褐色种皮候选基因GmF3’H的编码区及起始密码子上游1465 bp序列也是一致的。因此,证明褐色种皮J1265-2大豆与其衍生亲本黄色种皮大豆J1265-1为近等基因系,其控制褐色种皮的基因型与已报道的基因型不同。     

Inheritance of seed condensed tannins and their relationship with seed-coat color and pattern genes in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol–HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.     

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants

Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.     

A transgenic,visual screenable marker for soybean seeds

Most soybean cultivars produce buff colored seeds due to a seed coat specific siRNA mechanism. This phenomenon is specifically limited to the seed coat and produces a strong visual effect, thus, a strategy to evade the silencing was used to produce a maternal transgenic marker for soybeans. Expression of a rice chalcone synthase transgene with little DNA sequence homology to the soybean siRNAs resulted in dark colored seed coats. This phenotype is the result of anthocyanin pigment production and does not appear to affect other tissues. This novel approach for producing an easily scored transgenic marker for soybean will facilitate high-throughput screening and analysis of transgenic soybean.     

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.     

光、糖与激素影响植物花色素苷合成与积累的研究进展(综述)

次生代谢物质花色素苷存在于植物的叶片、花、果实和种子的表皮细胞的液泡中,是一类使这些器官呈现从红色到黑色等系列颜色的水溶性色素。其合成过程不仅受到基因的调控,还受多种因素影响。首先是光通过信号转导途径直接或间接地调节相关酶基因表达的过程;其次是糖,常与光相互作用协调控制花着色;激素也是影响花色素苷合成的一个重要因素,往往通过影响植物体内的代谢过程和植物基因的表达来影响花色素苷的合成和积累。本文综述近20年来该领域的研究进展。     

Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations

The I locus controls inhibition of anthocyanin accumulation in the epidermal cells of the soybean seed coat and affects abundance of PRP1, a proline-rich cell wall protein in the seed coat. Saline-soluble PRP1 is abundant in the developing seed coats of cultivar Richland (homozygous I, yellow), while it is significantly decreased in the pigmented isogenic mutant T157 (homozygous i, imperfect black). In this report, we examined soluble PRP1 in several cultivars containing alleles of the I locus which affect spatial distribution of pigmentation in the seed coat. We also characterized PRP1 in isolines with allelic variants of several other loci involved in seed coat pigmentation, including T and Im. The T gene is pleiotropic and affects both pubescence color and seed coat pigmentation and structure. Soluble PRP1 was abundant in the developing seed coats of lines with yellow seed (I or i ⁱ alleles) regardless of pubescence color, just as in Richland. Likewise, soluble PRP1 was decreased in pigmented seed coats (i ^k or i alleles) with grey (t) pubescence, as in T157. However, the total seed coat proteins were not extractable from pigmented seed coats with tawny pubescence (i, T genotypes) because they have proanthocyanidins that exhibit tannin properties. The dominant Im allele inhibits seed coat mottling (irregular patches of pigmentation) that occurs if plants are infected with soybean mosaic virus. PRP1 was 35 kDa in mottled (im) isolines and 34 kDa in non-mottled (Im) isolines. PRP2, which is expressed later in seed coat development and in the hypocotyl hooks of soybean seedlings, was also smaller in Im isolines. In summary, some of the anthocyanin mutations affect the quantity of soluble PRP1 polypeptides, while others correlate with structural changes in developmentally regulated proline-rich proteins.     

日本蛇根草花青素还原酶基因的克隆及其生物信息学分析

花青素还原酶(anthocyanidin reductase, ANR)是原花青素合成途径中的关键酶,同时花青素还原酶对植物花色苷的积累也发挥着重要作用。本研究以日本蛇根草为材料,以其转录组测序结果为基础,设计引物通过RT-PCR方法成功克隆得到日本蛇根草ANR基因完整的cDNA序列,利用生物信息学方法和工具对日本蛇根草ANR基因的功能结构域、生理和化学参数、亲/疏水性、信号肽、二级结构等进行预测和分析。结果表明,该基因cDNA全长为1 020 bp,编码339个氨基酸,预测其蛋白分子量为36.37 kD,等电点为5.92,为亲水蛋白,不含信号肽序列。综上,本研究成功克隆获得日本蛇根草ANR基因并完成其生物信息学分析,研究结果将为解析该基因的功能奠定基础。     

Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominant I alleles.

The seed of all wild Glycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (Glycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or i) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of near-isogenic isolines that vary in pigmentation as specified by combinations of alleles of the I, R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7- to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.     

Duplications That Suppress and Deletions That Restore Expression from a Chalcone Synthase Multigene Family

Seed coat color in soybean is determined by four alleles of the classically defined / (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the / locus, we demonstrated that the / locus is a region of chalcone synthase (CHS) gene duplications. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the / locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silencing and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant ii allele (yellow seed coats with pigmented hila) to the recessive i allele (fully pigmented) can be associated with the absence of a 2.3-kb Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutations exhibit deletions in the CHS4 promoter region. The dominant / allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridizes with both the CHS coding region and CHS1 promoter-specific probes. Mutations of the dominant / allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-kb Hindlll CHS fragment associated with the dominant / allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed in seed coats.     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.